1. Field of the Invention
The present invention relates to novel transglutaminase preparations derivable from the class Oomycetes, a novel transglutaminase derived from Phytophthora cactorum, CBS 618.94 or IFO 30474, a DNA construct encoding the transglutaminase enzyme, a method of producing the novel transglutaminase and the novel transglutaminase preparation, a method for producing a gel or protein gelation composition, and the use thereof.
2. Description of Related Art
Transglutaminases are enzymes capable of catalyzing an acyl transfer reaction in which a gamma-carboxyamide group of a peptide-bound glutamine residue is the acyl donor. Primary amino groups in a variety of compounds may function as acyl acceptors with the subsequent formation of monosubstituted gamma-amides of peptide-bound glutamic acid. When the ε-amino group of a lysine residue in a peptide-chain serves as the acyl acceptor, the transglutaminases form intramolecular or intermolecular ε-(γ-Glu)-Lys crosslinks.
This peptide crosslinking activity is useful for a variety of industrial purposes, including gelling of proteins, reduction of antigenicity of proteins, improvement of baking quality of flour, producing paste type food materia from protein, fat and water, preparation of cheese from milk concentrate, binding of chopped meat product, improvement of taste and texture of food proteins, producing jelly, gel cosmetics etc.
A wide array of transglutaminases have been isolated and characterized from animals and plants. The animal derived TGases are Ca2+-dependent and often multi-subunit enzymes. The most widely used mammalian transglutaminase, FXIIIa, is product inhibited, difficult to obtain in high amounts and thus expensive, and therefore not useful for all applications.
A few microbial TGases have been described, including the Ca2+-independent TGases from Streptoverticillia disclosed in U.S. Pat. No. 5,156,956 and related species disclosed in U.S. Pat. No. 5,252,469.
The yields of the microbial transglutaminases in shake-flasks and fermentors are far below those seen for other industrial enzymes. Thus, better production methods, including new high-yielding producers are needed. Previously, this goal has been pursued by applying conventional recombinant DNA techniques for cloning and expression in E. coli, S. cerevisiae and S. lividans (Washizu et al.; Tahekana et al.; Takagi et al.) but without success.
Klein et al. found and partially characterized a transglutaminase from the slime mold Physarum polycephalum which is a homodimer having a total molecular weight of 77 kDa. JP 6078783 Kokai relates to the use of this transglutaminase for protein gelation. However, it is well-known that slime molds are unsuited for large-scale industrial fermentation. Further, Physarum is not a fungus; it belongs to the Myxomycetes (Entrez NIH data base, current version January 1996). Taxonomically, the only common feature of Oomycetes, Myxomycetes and Eumycota (fungi) is that they all are mitochondrial eukaryotes.
The object of the invention is to provide a novel transglutaminase, a novel transglutaminase preparation, a method for producing the transglutaminase or transglutaminase preparation in a better yield and higher purity than hitherto possible which transglutaminase can be used either alone or in combination with other enzymes for industrial purposes.